Our protocol provides a way for us to study antibiotic-resistant bacteria in a nutritional and physical environment similar to how they likely exist in vivo. One of the greatest advantages of this technique is the ability to capture high-resolution images and phenotypic data of bacteria in infection-relevant population sizes. That's aggregates of approximately 10 to 1, 000 cells.
Demonstrating the procedure will be Alexa Gannon, a graduate research assistant from my laboratory. To begin, sterilize the mucin-containing medium under ultraviolet light for four hours. Transfer the UV-treated mucin into sterile 1.7 milliliter tubes under sterile conditions and store the tubes at minus 20 degrees Celsius.
After preparing the buffered base, add the stock solution of mucin into the buffered base containing DNA as described in the text manuscript. On the evening before the experiment, inoculate five milliliters of LB broth with several colonies of Pseudomonas aeruginosa PAO1 pMRP91 from an antibiotic containing LB agar plate and culture the bacteria overnight at 37 degrees Celsius with agitation at 250 revolutions per minute. The next morning, at least two hours before starting the experiment, turn on the confocal laser scanning microscope and open the incubation module in the associated imaging software.
Dilute 500 microliters of the culture in five milliliters of fresh LB broth and incubate the bacterial suspension at 37 degrees Celsius and 250 revolutions per minute for 60 to 90 minutes. When the bacteria have entered the log phase, pellet the bacterial cells by centrifugation and wash the cells three times with filter sterilized PBS. After the last wash, resuspend the pellet in one milliliter of PBS.
Measure the absorbance of the bacterial cell suspension at 600 nanometers to determine the culture volume required for an optical density of 0.05 in five milliliters of synthetic cystic fibrosis sputum medium two. Inoculate the calculated volume of bacterial cell suspension in the medium and vortex gently before adding one milliliter of cells to each chamber of a four-chambered optic culture dish, then incubate the bacteria for four hours under static conditions at 37 degrees Celsius. To image the Pseudomonas aeruginosa aggregates by confocal laser scanning microscope, at the end of the incubation, designate three wells of the culture plate as antibiotic treatment replicates and one well as the no treatment control and place the plate on the heated stage in the microscope.
Select a 63X oil immersion objective and open the locate tab to identify the bacterial aggregates in the brightfield. Define an area for imaging within each well and use the positions module to store the area position. Set the excitation wavelength to 488 nanometers and the emission wavelength to 509 nanometers.
In the acquisition module, select the Z-stack option to acquire the images and use the line averaging module to reduce the background fluorescence in the GFP channel within the total volume of the Z-stack images. Set the time series option to capture 60 slices in each well at 15-minute intervals for 18 hours and use the definite focus strategy to store a focal plane for each position that will be re-imaged at each time point throughout the experiment. 4-1/2 hours after setting up the imaging experiment, image each position to determine the aggregate biomass within each of the four wells before the addition of the antibiotic.
After six hours, gently add antibiotic at two times below the minimum inhibitory concentration to the middle of each well just blow the air liquid interface and click start experiment to begin the post-antibiotic treatment imaging. To isolate the live cells, at the end of the 18-hour imaging period, label the bacteria in each well with the appropriate volume of propidium iodide according to the manufacturer's recommendations. At the end of the incubation, use an insulated container to transfer the plate to the flow cytometer and set the cytometer to detect GFP and propidium iodide using a 70 micron nozzle, then run one milliliter aliquots of each culture supernatant at the lowest flow rate to sort the viable GFP-positive and non-viable propidium iodide negative Pseudomonas aeruginosa aggregates.
To quantify the aggregate dynamics, load the images into an appropriate image analysis software program and create histograms of the counts produced for the untreated control and antibiotic-treated cultures in the GFP channel to allow quantification of the background fluorescence. To ensure that the detected GFP voxels correlate to the bacterial biomass, define a GFP-positive voxel as greater than or equal to 1.5 times the GFP background count value. Produce the ISO surfaces for all of the remaining voxels and to detect individual aggregates, enable the split objects option and define the aggregates as objects.
Use the vantage module to calculate the volume XYZ and sum of GFP voxels for each individual object. Export this data to an external statistical platform. Filter the exported data by size to define objects with volumes greater than or equal to five cubic micrometers.
Remove any objects less than 0.5 cubic micrometers and use the vantage module to calculate the distance of each object in relation to other objects within each image. Alternatively, the distance can be calculated using the formula, then use the sum and average calculations to determine the total biomass in the average aggregate volume. Although multiple viable bacterial aggregates remain after four hours of antibiotic application, the total biomass of the aggregate populations within the antibiotic-treated cultures is significantly reduced compared to that of untreated cultures.
Time series microscopy can be used to determine the spatial patterns between sensitive propidium iodide positive and tolerant GFP-positive aggregates after antibiotic treatment. By calculating how aggregates of different sizes contribute to the overall population, patterns of how aggregates respond to antibiotic treatment in relation to their size, shape, and position can be identified. After antibiotic treatment, viable and non-viable aggregates can be successfully separated according to their fluorescent signal expression.
We hope this protocol inspires other researchers to study their organism of choice at a similar resolution. Our goal is to find common biological threads in these complex communities regardless of the infection site.
